Plasmid purification with Qiagen’s Maxi Kit consists of a modified alkaline lysis procedure, followed by binding of the plasmid to Qiagen resin (in a pre-packed column) under low-salt conditions. The column is then washed and the plasmid DNA eluted with medium-salt and high-salt buffers, respectively. The Qiagen Plasmid Maxi Kit is designed to perform purification of plasmids up to approximately 150 kb, with possible reduced elution efficiencies for constructs larger than 45–50 kb. Qiagen Plasmid Maxi Kit has been developed to purify either high- or low-copy plasmids and cosmids. When purifying very low-copy plasmids and cosmids, the purification efficiency can be improved by washing the bacterial pellet in 20 ml 10.3% sucrose prior to adding lysis buffer, and also precipitating the DNA with 0.7 volumes of isopropanol with 2 ìL of glycogen.
I use the Qiagen Plasmid Maxi Kit to purify various types of plasmids, low and high copy plasmids that vary in size from 3 kb to 6 kb. The plasmids are usually transformed into DH5á cells and then grown in 200 to 400 mL of regular LB media over a period of 10 to 15 hours.
After pelleting the cells via centrifugation, the cells are resuspended in Buffer P1. I use the optional LyseBlue color indicator, added to the resuspension buffer (Buffer P1) before use. This provides visual identification of optimum buffer mixing (following addition the additional buffers for cell lysis and neutralization) preventing common handling errors that lead to inefficient cell lysis and incomplete precipitation of SDS, genomic DNA, and cell debris. I also recommend using the QIArack to hold the columns during the various column washes and elutions. The rack is very convenient and less risky than other systems put together with random equipment found in the lab.
The Qiagen Plasmid Maxi Kit is efficient and reliable. Every time I use this product, I obtain satisfactory amounts of purified plasmid DNA. The protocol is easy to follow and the entire purification process is very easy to perform. Moreover, the handbook gives valuable explanations on the different steps of the process, which allow you to optimize each step and improve purifications.
The whole purification process is quite long: from 4 hours to a whole day, depending on the number of samples to purify. The amount of time spent waiting for centrifugations, washes, or elutions varies from 15 and 40 minutes; this is not enough time to undertake other tasks during the purification process. Thus, 1 day of work can be entirely dedicated to the purification of plasmids.
Finally, like the majority of biology kits, the volumes of provided products are not in accordance with the protocol recommendations, resulting in left over product that will be inevitably wasted.